Centrifugation utilizes the principle that constituents (e.g., cells, cell clusters) within a medium (e.g., liquid solutions/mixtures) will assume a particular radial position within the centrifuge bowl based upon their respective densities and will therefore separate when the centrifuge is rotated at an appropriate angular velocity for an appropriate period of time. As can be appreciated, there are a variety of centrifuges having features which potentially enhance and/or restrict their use for particular applications. For instance, the COBE 2991.TM. Cell Processor is a variable volume centrifuge which is commercially available from the assignee of this patent application and is the subject of U.S. Pat. No. 3,737,096 to Jones et al., issued Jun. 5, 1973. This variable volume centrifuge has a variety of desirable features, including providing a centrifugation system which enhances the potential for maintaining a desired degree of sterility in various of the aspects involved in/relating to the actual separation of such constituents from the medium, such as during the provision of the medium to the centrifuge bowl and during the removal of the separated constituents from the centrifuge bowl.
Generally, a variable volume centrifuge has a substantially flexible diaphragm which separates the centrifuge bowl into upper and lower chambers. A substantially flexible processing bag is positionable within the upper chamber and is used to contain the medium to be centrifuged therein, as well as other appropriate additives. This processing bag is interconnectable with a plurality of sources and collectors via tubing and a centrally located rotating seal. The lower chamber of the centrifuge bowl is used to vary the volume of the upper chamber, and thus the processing bag, to allow for the on-line, sequential harvesting of fractions (i.e., a certain volume of medium) from the processing bag.
In preparation for centrifuging, the processing bag is positioned within the upper chamber of the centrifuge bowl and is interconnected via the rotating seal and tubing to the sources and collectors. The medium to be centrifuged is typically provided to the processing bag after the bag is positioned within the centrifuge bowl. The potential for maintaining a desired degree of sterility is enhanced by providing such medium from at least one of the sources to the processing bag via the tubing (i.e., no additional exposure of the medium is required when providing such to the processing bag). When rotation of the centrifuge is thereafter initiated, the various constituents begin to assume a particular radial position within the centrifuge bowl based upon their respective densities. When appropriate separation has been achieved, a hydraulic or other appropriate fluid is provided to the lower chamber of the centrifuge bowl during continued rotation of the bowl to raise the diaphragm and thus reduce the volume of the upper chamber and the flexible processing bag contained therein. As a result, fractions are sequentially removed from the processing bag via the rotating seal and tubing by order of increasing density and at least one of such fractions will have the desired separated constituents therein. These various fractions may be directed to one or more of the collectors, either for storage and subsequent use and/or for disposition as waste.
Swinging bucket-type centrifuges are also commercially available, one of which is the Mistral 300I by MSE. Generally, the material to be centrifuged is placed in a centrifuge tube having a closable top and is thereafter secured to a radially-extending arm within the centrifuge. A plurality of these radially-extending arms may be utilized by a given centrifuge (typically ranging from a 2 tube capacity to a 25 tube capacity) to increase processing capabilities since the tubes are of generally small volume (e.g., 50 milliliters). Once the centrifuge tubes are appropriately positioned, the centrifuge is rotated such that the arms with the tubes attached thereto swing out to a position determined by centrifugal force. Usually, the tubes assume a substantially horizontal position. Upon rotation of the centrifuge at an appropriate angular velocity and for an appropriate period of time, separation is achieved within each tube based upon the density of the various constituents. Thereafter, the rotation of the centrifuge is terminated, typically by allowing for a free spinning of the centrifuge to the static state to attempt to reduce the potential for disturbing the achieved degree of separation to an undesirable degree. Moreover, this also allows the tubes to return to their initial position. Once rotation of the centrifuge has stopped, the centrifuge tubes are removed therefrom and the desired separated constituents are harvested from the tube, typically by passing a syringe through the top of the tube to withdraw fractions therefrom until the desired separated constituents are removed and collected.
One particular application suited for use of the above-described principles of centrifugation is the harvesting of various types of cells and/or cellular structures from cell mixtures, such as islets of Langerhans from a preparation of a pancreatic digest having such islets and exocrine tissues therein. These harvested islets are useful in the treatment of diabetes by transplantation of the islets or otherwise. In this case it is highly desirable for the centrifugation to produce a high degree of separation, and thus highly-purified islets. For instance, possible advantages associated with transplanting highly-purified islets include increased safety (e.g., reduced health risks), improved islet implantation, and reduced immunogenicity of the graft. N. London, R. James, & P. Bell, "Islet Purification" in Pancreatic Islet Cell Transplantation, Camillo Ricordi, ed. 113 (1992).
One available alternative for the separation of islets from a preparation of a pancreatic digest is by utilizing centrifugation in which, generally, separation is primarily based upon cell density. One type of such centrifugation is centrifugation in a discontinuous density gradient, and these gradients have been utilized in the harvesting of islets both in variable volume centrifuges and swinging bucket type centrifuges of the above-described types.
Separation by centrifugation utilizing a discontinuous density gradient will be explained herein by reference to a swinging bucket type centrifuge and generally entails the use of a plurality of solutions having known but differing densities such that a pre-established, vertically layered configuration is metered into a tube 20 as illustrated in FIG. 1. In this case, a discontinuous density gradient 22 is formed by first, second, third, and fourth solutions 24, 28, 32, 36 which have different densities, the densities of the various solution layers increasing in moving down the gradient 22 as indicated by the spacing changes in the solution designations (i.e., the density of the first solution 24 is less than the density of the second solution 28 and so forth). By selecting appropriate density solutions for the gradient 22, the desired cell(s) to be harvested will collect at least at one of the three interfaces 40, or at the top or bottom of the tube 20, during rotation of the centrifuge (swinging bucket type) in which the tube 20 is positioned in the above-described manner, whereas remaining portions of the cell mixture will collect at other interfaces 40 within the gradient 22. Therefore, the desired cells will separate from remaining portions of the cell mixture during centrifugation and can be harvested by removing the tube 20 from the centrifuge and drawing the desired cells off in a syringe in the above-described manner.
As noted above, instead of having the discontinuous density gradient 22 within the tube 20, in the case where a variable volume centrifuge of the above-described type is utilized the gradient 22 may be provided to its flexible processing bag and be radially extending therein. This again allows for on-line, sequential removal of fractions to obtain the desired cells which collect at one or more of the interfaces 40 which would be substantially annular and positioned at varying radial distances from the rotational axis of the centrifuge bowl.
Cell separation by utilizing a discontinuous density gradient has a number of disadvantages. For instance, the operative part of the gradient is the zone at each interface 40 within the gradient 22. More particularly, the various solution densities are selected such that the desired cell will pass through, for instance, the first and second solutions 24, 28 (i.e., the cells will have a greater density than each of these solutions), but will have a density which is less than that of the third solution 32 such that the cells will collect at the interface 40 between the second and third solutions 28, 32. Furthermore, the other constituent(s) of the mixture containing such cells will, on the other hand, have for instance a density such that these constituents will pass through the first, second, and third solutions 24, 28, 32, but will have a density less than that of the fourth solution 36 such that these constituents will collect at the interface 40 between the third and fourth solutions 32, 36. Based upon this separation technique, it can be appreciated that the interfaces 40 may become occupied by cells during centrifugation to the degree where such cells will act as a barrier to the movement of other constituents within the cell mixture toward a more radially outwardly positioned interface 40, particularly in the case where the material to be centrifuged is "top loaded" onto the gradient 22. "Top loading" is where the material to be centrifuged is provided to the tube 20 after the gradient 22 is in the tube 20. Consequently, this effectively limits the amount of cell mixture which may be processed utilizing a discontinuous density gradient.
Related to the foregoing clogging problem at the interfaces 40 often associated with discontinuous density gradients 22 is that an overloading of the gradient 22 may introduce a certain instability to the gradient 22 which may adversely affect separation. Moreover, in the event that the cells and other constituents of the cell mixture have relatively close densities, the potential increases for such clogging at the interfaces 40 of a particular density, and thus may result in a reduction in the purity of the cells to be harvested. Moreover, in order to enhance the effectiveness of the separation by a discontinuous density gradient 22, the individual gradient layers should preferably be selected based upon certain criteria since the characteristics of the cells and/or cell mixture may vary depending upon, for instance, the donor/source and/or the manner in which the cell mixture is obtained. Further, the characteristics of the cells obtained from a given donor or source may vary.
Another type of gradient which may be utilized in separation by centrifugation is a continuous density gradient in which the density changes continuously throughout the gradient solution (e.g., there are infinitesimal interfaces) such that the cells effectively migrate to their respective buoyant densities within the gradient solution. This has been utilized in the above-described types of swinging bucket centrifuges having the substantially rigid wall centrifuge tubes for containing the gradient solution and the material to be separated by centrifuging. One particular application in which a continuous gradient has been utilized is in the separation of DNA and RNA molecules as illustrated in FIG. 2. In this case, a gradient solution (not yet established) is provided to the tube 44, as well as the solution containing the DNA/RNA molecules. In this particular application, the limited volumetric capacity of the tube 44 is tolerable since the amount of DNA and RNA molecules which may be collected in this tube 44 is sufficient for the intended uses of such molecules.
The continuous gradient 48 is established within the tube 44 by rotating the swinging bucket-type centrifuge at a sufficiently high velocity and for an extended period of time such that the gradient 48 is establishes within the tube 44 (e.g., 45,000 RPM for 36 hours). As a result, the DNA and RNA molecules each migrate to their respective positions within the gradient solution based upon their particular densities. In this regard, the DNA molecules collect at first and second regions 52, 56, whereas other portions of the mixture such as the protein migrate further within the gradient solution to form a third region 60. The RNA may actually pass completely through the gradient 48 as illustrated by a pellet 64.
A continuous gradient of the above-described type has also been utilized in the selection of the particular gradient solution layers (e.g., solution densities) to be used in a discontinuous density gradient separation method utilizing a variable volume centrifuge since as noted above it is desirable to adapt each gradient to certain characteristics. Moreover, a continuous gradient has been used in a centrifuge in which the centrifuge tube is maintained in a substantially vertical position such that the gradient is radially established therewithin (i.e., horizontally extending). When appropriate separation has been achieved in this particular case, the centrifuge tube is removed from the centrifuge and the gradient actually inverts such that it extends vertically within the centrifuge tube. The desired fraction(s) may thereafter be harvested from the centrifuge tube in the above-described manner.